Method and compositions for treating respiratory pathologies

ABSTRACT

The present application relates to compositions and methods for treating respiratory pathologies. It equally concerns compositions and methods allowing regulation of the paracellular permeability of the pulmonary epithelium. The compositions and methods of the invention are based in particular on the use of agents or conditions modulating the tension of the cytoskeleton of pulmonary epithelial cells, particularly enterocytes. The invention may be used for preventive or curative treatment of various pathologies, such as asthma, allergies, obstructive diseases, etc., in mammals, particularly humans.

The present application relates to compositions and methods for treatingrespiratory pathologies. It equally concerns compositions and methodsallowing regulation of the paracellular permeability of the pulmonaryepithelium. The compositions and methods of the invention are based inparticular on the use of agents or conditions modulating the tension ofthe cytoskeleton of pulmonary epithelial cells. The invention may beused for preventive or curative treatment of various pathologies, suchas asthma, allergies, obstructive diseases, etc., in mammals,particularly humans.

The pulmonary epithelium is the site of very important exchanges betweenthe external environment and the body. These exchanges can take placeeither across the cells of the epithelium, or by parallel systems. Forinstance, the transport of water or electrolytes, or yet the absorptionof small molecules (molecular weight generally less than about 1000 Da)in the gastric, intestinal or colonic mucosa, takes place by thetranscellular route, across epithelial cells or enterocytes. Incontrast, the absorption of large molecules and the passage of toxins orimmune cells occurs principally by the paracellular route, at the levelof “tight junctions”, which are located between epithelial cells.

Epithelial tight junctions (or “TJ”) are linker structures between thecells lining the mucosal epithelia (gastrointestinal tract, lungs).These structures ensure and control paracellular transepithelialtransport, from the exterior towards the submucosa, of variousmacromolecules (irritants, microorganisms). These structures also enablethe migration of immune cells (e.g., immunocytes) towards the exterior.Tight junctions are flexible structures composed of a complex assemblyof transmembrane proteins (occludins, claudins) and cytoplasmic proteins(zona ocludens proteins Z0-1, ZO-2, ZO-3, AF7 proteins, cingulin or 7H6,etc.), which are associated with the components of the cytoskeleton(myosin, actin filaments, etc.). Moreover, agents that disrupt actincytoskeletal organization have been found to upregulate endothelial cellNitric Oxide Synthase activity (WO 00/03746).

Under physiologic conditions, the degree of “partial” opening of thesetight junctions permits the local immune system to be informed of thenature or “quality” of the contents of the airways.

While the intestinal epithelium and the structure of its tight junctionshave been studied in the prior art, at present there are fewer dataconcerning the pulmonary epithelium and the functioning of its tightjunctions.

The process of sensitization to allergens is a very important riskfactor in the development of allergies and asthma. However, themechanisms by which allergens initiate the phenomenon of sensitizationhave not been clearly documented in vivo. When allergens are inhaled,they come into contact with the pulmonary epithelial wall which preventsthem from entering the body and presenting to immune cells. Nonetheless,for sensitivity to develop, this implies that certain allergens must beable to cross this epithelial barrier to interact with immune cells. Theconditions under which this transfer is possible have not been clearlyelucidated in vivo. Thus, while some in vitro studies in cell culturessuggest a role of tight junctions in this process, there are no in vivodata on the role of these junctions in the development of sensitization.Likewise, although Gordon et al. (Exp. Lung Res. 1998; 24: 659) suggestthat taurine has a protective effect on tight junctions, there are nodata establishing a correlation between tight junctions andsensitization or transfer of allergens across the pulmonary epithelium.

Experimental studies (WAN, H et al. J. Clin. Invest, 1999; 104(1):123-33) as well as the demonstration in asthmatic subjects of acorrelation between the size of extracellular spaces and the respiratoryresponse threshold to inhaled acetylcholine (OHASHI et al. Aerugi 1990November; 39(11): 1541-5) suggested a correlation between the degree ofopening of tight junctions and the response to airborne allergens.However, these preliminary findings were not confirmed and did not giverise to new therapeutic approaches.

The present application results from the demonstration of an in vivorole of pulmonary epithelial tight junctions in the allergensensitization process. The present application also follows from thedevelopment of new therapeutic strategies for treating respiratorypathologies, based on modulating the paracellular permeability of thepulmonary epithelium. In particular, the present application proposes,for the first time, a therapeutic approach to respiratory pathologiesbased on the use of compounds or conditions allowing modulation of thetension of the cytoskeleton of pulmonary epithelial cells. Inparticular, this approach allows control of the opening and closing ofpulmonary epithelial tight junctions, without necessarily requiring denovo protein synthesis and/or significant protein and/or structuraldegradation in the epithelium. Such strategy allows the permeability ofthe pulmonary epithelium to be regulated in a specific, subtle andreactive manner, and thus to act on the transfer of allergens towardsthe immune cells. This strategy is especially suited to obtaining arapid biological effect controllable over time (reversible).

In this respect, the results presented herein show that a substance ableto relax epithelial tight junctions (receptor activator peptide RAP-2,LPS) promotes the accumulation of neutrophils and eosinophils in thepulmonary alveoles, as observed in bronchopulmonary disorders such asasthma. The results obtained further show that a chemical substance ableto reduce the permeability of tight junctions of the pulmonaryepithelium prevents the accumulation of neutrophils and eosinophils.These findings offer proof that molecules, agents, conditions or methodsable to reduce or suppress the opening of tight junctions of thepulmonary alveolar or bronchial epithelium may be of value in treatingpulmonary disorders, particularly those characterized by intrabronchialand alveolar accumulation of neutrophils and eosinophils, in particularasthma.

A first object of the invention is therefore based on a compoundmodulating the tension of the cytoskeleton of pulmonary epithelialcells, for preparing a medicament intended for the preventive orcurative treatment of respiratory pathologies, preferably with theexclusion of hypoxia induced by respiratory pathologies, in particularimpaired lung function. In that respect, impaired lung function can becaused by emphysema, cigarette smoking, chronic bronchitis, asthma,infection agents, pneumonitis (infectious or chemical), lupus,rheumatoid arthritis, inherited disorders such as cystic fibrosis,obesity, α₁-antitrypsin deficiency and the like. Hypoxia as used hereinis defined as the decrease below normal levels of oxygen in a tissue.

Another object of the invention is directed to a method of preventive orcurative treatment of respiratory pathologies, comprising administeringto a subject in need of such treatment an effective quantity of acompound modulating the tension of the cytoskeleton of pulmonaryepithelial cells.

The invention is thus based on the use of compounds modulating thetension and the state of contraction of the cytoskeleton of pulmonaryepithelial cells. As indicated hereinabove, this approach enablescontrol of the opening and closing of pulmonary epithelial tightjunctions, without necessarily requiring de novo protein synthesisand/or significant protein and/or structural degradation in theepithelium.

The proteins composing the tight junctions are associated with thecytoskeleton of the cells they link together. It is proposed within thecontext of the invention that the tension of the cytoskeleton can bemodulated in subjects presenting with respiratory disorders or diseasesso as to act non-destructively and transiently on the permeability oftheir pulmonary epithelium. For example, contraction of the cytoskeletonshould promote the opening of tight junctions, whereas relaxation of thecytoskeleton (or inhibition of contraction) should promote closing ofthe tight junctions.

Within the scope of the invention one therefore preferably usescompounds (or conditions) that modulate the contraction of thecytoskeleton of pulmonary epithelial cells (particularly human),preferably without substantially modulating the endothelial vascularand/or circulating hemodynamic permeability. Depending on the conditionto be treated, one uses compounds which inhibit the contraction of thecytoskeleton of pulmonary epithelial cells, or which activate or promoteit.

The activity of the compound on cytoskeletal tension may be direct orindirect, that is to say directed on the cytoskeletal componentsthemselves or on components that regulate its tension. Although notlimiting, compounds acting directly on the tension of the cytoskeletonare preferred. Furthermore, also preferred are compounds having aselective activity on the tension of the cytoskeleton, that is to saytypically compounds which do not directly affect the structure of thecomponent proteins of the tight junctions.

A compound is considered to modulate the tension of the cytoskeletonwhen it modulates the opening of tight junctions. Inhibition ofcontraction does not necessarily have to be complete or total, butcontraction must be reduced sufficiently to reduce the opening of tightjunctions such that the minimum decrease in paracellular permeability ofthe pulmonary epithelium is approximately 30%, preferably approximately40%, even more preferably approximately 50%.

Different types of compounds may be used within the scope of the presentapplication. Thus, according to the invention, the term “compound” mustbe interpreted in the broad sense, i.e. as designating any agent,substance, composition, condition, treatment or method allowingmodulation of cytoskeletal tension. In an advantageous manner it is anagent (e.g. a molecule) or a combination or association of molecules.

According to a first preferred embodiment, one uses compounds whichinhibit (or modulate) the contraction of the myosin light chain, orcompounds which inhibit (or modulate) the degradation of actin.

An especially preferred embodiment of the invention consists in the useof compounds which inhibit (or modulate) the contraction of the myosinlight chain or the degradation of actin.

In a particularly preferred embodiment, the invention is implemented byusing compounds that inhibit the contraction of the myosin light chainor the degradation of actin, in particular compounds that inhibitphosphorylation of the myosin light chain.

Such compounds may be exemplified in particular by inhibitors of themyosin light chain kinase (MLCK).

A particular example of such selective (MLCK) inhibitor is compound ML-7{1-(5-iodonaphtalène-1-sulfonyl)-1H-hexahydro-1,4-diazepine} (MakishimaM. et al. FEBS Lett. 1991; 287:175). Other examples of such inhibitorscan be cited such as compound ML-9 (Wilson DP. et al. J. Biol. Chem.2001;13: 165) or other which are non selective: Wortmannin (Warashina A.Life Sci 2000;13: 2587-93), H-7 (Piao Zf et al. Mol Cell Biol Res Commun2001;4: 307-12) et KT 7692 (Warashina A. Life Sci 2000;13: 2587-93). Aparticular object of the present invention is the use of compounds thatinhibit MLCK selected in the group consisting of ML-7, ML-9, Wortmannin,H-7 and KT 7692, which may be alone or in combination thereof. Preferredcompounds of the invention are compounds that do not present asignificant or substantial effect on the vascular and/or pulmonarycirculating hemodynamic permeability. In a particular embodiment, thepresent invention comprises the use of compounds that inhibit MLCK byexcluding compounds selected in the group consisting in BDM[2,3-butanedione 2-monoxime], ML-7 [1- (5-iodonaphthalenel-sulphonyl)-1H-hexahydro-1,4-diazepine hydrochloride], ML-9 [1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepinehydrochloride], wortmannin, H-7 [1- (5-isoquinolinesulphonyl)-2-methylpiperazine dihydro-chloride], Fasudil (HA1077)[Hexahydro 1- (5-isoquinolinesulphonyl)-1H-1,4- diazepine], W-7[N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide] and A-3[N-(6-Aminoethyl) -5-chloro-1-naphthalenesulfonamide]. Other compoundsthat inhibit phosphorylation of the myosin light chain can be compoundsthat activate the myosin phosphatase.

Other targets acting on the tension of the cytoskeleton are notablymyosin binding proteins, such as for example cingulin, or junctionmolecules, such as cadherin-E, catenin-α or desmosomes. Modulation ofthe activity or the expression of these proteins permits regulation ofcytoskeletal tension, within the scope of the present invention.

A specific object of the invention is therefore directed to the use of amodulator (particularly an inhibitor) of the activity or the structureor the expression of molecules of the cytoskeleton. For example, thecompound may be an antisense nucleic acid, a synthetic molecule, anantibody fragment, etc.

According to another embodiment, compounds may be used which inhibit thesynthesis of proteins or other molecules ensuring the link between theproteins of the cytoskeleton and the proteins of the tight junctions.Among the tight junction proteins may be cited in particular theproteins occludins, claudins, ZO-1, ZO-2, ZO-3, AF7 and 7H6. Theinvention provides a means of modulating the opening or closing of tightjunctions which is therefore based on regulating the synthesis of linkerproteins between the cytoskeleton and the proteins of the tightjunctions. By stimulating such synthesis, a reinforcement of the linkbetween tight junctions and the cytoskeleton is expected, leading todecreased permeability of the epithelium.

Other compounds that may be used in the invention comprise for exampleinhibitors of mitogen activated kinases (MAPKK), particularly the kinaseMEK1 or kinase P13, such as compounds PD098,059{2-(amino-3-methoxyphenyl)-4H-1-benzopyran-4-one} (Alessi et al., J.Biol. Chem. 1995; 270, 27589) or LY294002 {2-(4-morpholinyl)-sphenil-1(4H)-benzopyran-4-one} (Vlahos et al., J. Biol. Chem. 1994; 269: 5241).

Other molecules that may be used to indirectly regulate the tension ofthe cytoskeleton include growth factors, such as hepatic growth factor(HGF), endothelial growth factor (EGF) or certain cytokines that can bereleased by immune cells, such as interleukins-1, -4, -13, or factorssuch as IFG-1 or gamma-interferon.

Another approach for indirectly regulating the tension of thecytoskeleton is based on the use of taurine or the peptide GLP2(glucagon-like peptide 2) or yet derivatives thereof, which can alterpulmonary epithelial permeability through an indirect effect oncytoskeletal contraction. Similarly, certain molecules acting on thereceptors located at the apical pole of epithelial cells (e.g.,proteinase receptors; PAR-2) can act indirectly on the cytoskeleton.

A preferred embodiment of the invention comprises the use of agentsacting directly on the tension of the cytoskeleton, particularlymolecules which inhibit cytoskeletal contraction, especially moleculeswhich inhibit the contraction of the myosin light chain, or whichinhibit the degradation of actin.

As noted hereinabove, in an advantageous manner the compounds used aremolecules, which may be alone or in combination, biological extracts,etc. Such molecules may be synthetic, semi-synthetic or biological,particularly of animal, viral, plant or bacterial origin.

The present invention may be used for treating or managing diseases ordisorders of the respiratory system, particularly asthma, allergies,obstructive disorders (bronchitis, bronchiolitis, emphysema, etc.),especially when such pathologies are chronic or severe. It isparticularly adapted to the preventive or curative treatment of asthmaor various allergies (dust, pollen, pollution, etc.) as well as to thelocal treatment of lung inflammation. It may be used preventively insubjects with a predisposition or sensitivity to this type of disorder,or curatively, for example during attacks or over longer periods. Thecompositions and methods of the invention make it possible to reduce thesuffering or respiratory difficulties of subjects, and attenuate thesymptoms or the cause of these disorders.

A particular object of the invention is based on the use of a compoundsuch as defined hereinabove for preparing a medicament intended tocontrol, particularly to reduce, the paracellular permeability of thepulmonary epithelium of subjects with respiratory diseases, particularlypulmonary disorders characterized by intrabronchial and alveolaraccumulation of neutrophils and eosinophils, for example asthma andallergy.

Another particular object of the invention consists in the use of acompound such as defined hereinabove for preparing a medicament intendedto reduce sensitization to allergens in subjects presenting with orsensitive to respiratory diseases, particularly pulmonary disorderscharacterized by intrabronchial and alveolar accumulation of neutrophilsor eosinophils, for example asthma or allergy.

A further particular object of the invention consists in the use of acompound such as defined hereinabove for preparing a medicament intendedto reduce transepithelial migration of immune cells and accumulation ofimmune cells in the lungs of subjects presenting with a respiratorydisease, particularly a pulmonary disorder characterized byintrabronchial and alveolar accumulation of neutrophils or eosinophils,for example asthma or allergy.

The invention equally relates to methods for treating the hereinaboveconditions, comprising administering to a subject presenting with arespiratory pathology or sensitive to respiratory pathologies, acompound or treatment such as defined hereinabove. In a preferredmanner, the compound or treatment is administered at an effective doseto reduce the paracellular permeability of the pulmonary epitheliumand/or to reduce sensitization to allergens and/or to reducetransepithelial migration of immune cells and accumulation of immunecells in the lung.

The compound may be administered by different routes and in differentforms. For example, the compound may be a liquid, solid or aerosol,typically in the form of a tablet, capsule, aerosol, ampoule or oralsolution, solution for injection, etc. Compounds formulated for localadministration are preferred (e.g. in the airways (e.g. respiratory) orby the oral route (oral solutions, tablets, ampoules, syrups, sprays,etc.). Aerosol packaging is especially preferred, when this is possible.Of course, other forms of administration are possible, such asinjections (intradermal, subcutaneous, intramuscular, intravenous,intra-arterial, intraperitoneal, etc.), ointments, gels, suppositories,etc.

The compounds may be used alone or in combination and/or in associationwith other active agents, such as for example other active substancesused in the treatment of respiratory diseases. Examples includeβ2-agonists and anticholinergics, corticosteroids, anti-leukotrienes,etc. These different agents may be used in multidrug therapy, andadministered separately, in combination, spread out over time orconcomitantly.

Another object of the invention is directed to a product or apharmaceutical combination comprising at least one compound thatmodulates the tension of the cytoskeleton of pulmonary epithelial cellsand at least one other active agent selected from among β2-agonists,anticholinergics, corticosteroids and anti-leukotrienes, in view ofcombined use, separate use or spread out over time.

A further object of the invention is a pharmaceutical compositioncomprising at least one compound that modulates the tension of thecytoskeleton of pulmonary epithelial cells according to the presentinvention, preferably a compound that inhibits the contraction of themyosin light chain, more preferably a compound that inhibits thephosphorylation of the myosin light chain, especially an inhibitor ofMLCK or an activator of the myosin phosphatase, and a pharmaceuticallyacceptable excipient, said composition being formulated preferably fororal administration or inhalation. Preferably, the compound isformulated as an aerosol and contains a carrier gas, or as an oralsolution.

The compound that modulates cytoskeletal tension of pulmonary epithelialcells used as the pharmaceutical active principle is employed intherapeutically effective amounts. It is understood that theadministered dose may be adapted by those skilled in the art accordingto the subject (patient) to be treated, the pathology concerned, themethod of administration, etc. The quantities or doses of the compoundsadministered or used in the compositions according to the invention maybe determined according to their capacity to modulate the cytoskeletaltension of pulmonary epithelial cells. This capacity and thereforesetting the dose to be administered may in particular be determined bythe experimental protocol described in example 7.

Other aspects and advantages of the present invention will becomeapparent in the following examples, which are given for purposes ofillustration and not by way of limitation.

LEGENDS OF FIGURES

FIG. 1: Effect of ML-7 on the increase in pulmonary paracellularpermeability to ¹²⁵I labelled human serum albumin induced byintratracheal infusion of Pseudomonas aeruginosa LPS. LPS decreasesradioactivity levels measured in the bronchoalveolar lavage fluid (BAL)whereas, in comparison with controls, these levels are significantlyhigher in the lungs. This increase in pulmonary permeability isinhibited by pretreating the animals with ML-7. In fact, radioactivitylevels in both BAL and lung in ML-7-treated animals were similar tothose of controls.

FIG. 2: Western blot of the phosphorylated (p-MLC) and native (MLC)myosin light chain following treatment of cultured NCI-H292 humanbronchial cells with LPS. Incubation times are shown on each blot(C=control).

EXAMPLES Example 1 Reduction of the Bronchial Inflammatory Response byTaurine

The bronchial and alveolar epithelium possesses structures linkingepithelial cells which allow controlled passage of immune cells into theairways. This example shows that certain molecules known to increaseintestinal paracellular permeability such as SLIGRL promote theintra-alveolar accumulation of immune cells (neutrophils, macrophages)and that this effect can be prevented (e.g., inhibited or reduced) byoral treatment with taurine.

For this experiment, four groups of 8 male Wistar rats (250-300 g) weregiven drinking water containing (groups 1 and 2) or not containing(groups 3 and 4) 5% taurine, for a period of 10 days.

At time t=10 days, the four groups of animals were given a slowintranasal instillation of 200 μl of physiologic serum containing(groups 2 and 4) or not containing (groups 1 and 3) 0.2 mg of SLIGRL.

At time t=3 h after intranasal instillation, animals were anesthetizedfor bronchoalveolar lavage, then sacrificed.

The results are given in Table 1 below.

These results show that intranasal instillation of SLIGRL results inaccumulation of eosinophils and neutrophils in bronchoalveolar lavagefluid (BAL) at t=3 h in control animals but not in animals treated withtaurine (Table 1). These results provide in vivo confirmation of therole of tight junctions in the permeability of the pulmonary epitheliumto immune cells. TABLE 1 Effect of taurine on neutrophil and eosinophilaccumulation in bronchoalveolar lavage fluid induced by intranasalinstillation of SLIGRL in rats (mean ± SD; n = 10) PAR 2 (0.2 mg/kg IN)Taurine 10% + PAR 2 (mean ± SEM 0.9% NaCl PAR 2 0.9% NaCl PAR 2 Tot. 960± 112 6464 ± 99⁺ 1728 ± 111 2086 ± 134* Leucocytes (mm³) Macrophages 945± 25  5559 ± 63⁺ 1651 ± 72  1967 ± 103* (mm³) Neutrophils   4 ± 0.3  656± 41⁺ 34 ± 3 34 ± 3* (mm³) Eosinophils 0.2 ± 0.7  32 ± 2⁺  17 ± 1 17 ±1* (mm³) Lymphocytes 144 ± 11   297 ± 28  22 ± 8 22 ± 8* (mm³)*p < 0.05 from PAR 2 values;⁺p < 0.05 from NaCl values;IN: intranasal

Example 2 Reduction of the Bronchial Inflammatory Response by ML-7

This example demonstrates that ML-7 reduces the intra-aveolaraccumulation of immune cells observed after an intratracheal infusion oftaurocholate which induces opening of tight junctions.

For this experiment, three groups of 8 male Wistar rats (250-300 g) weregiven either ML-7 by the IP route at a dose of 1 mg/kg/12 h for 36hours, or the vehicle alone. One hour after the last injection, a slowintratracheal instillation of 200 μl of physiologic serum containing(two groups) or not containing (control group) 50 mM taurocholate wasgiven.

At time t=2 h after the intratracheal instillation, animals wereanesthetized for bronchoalveolar lavage, then sacrificed.

The results are presented in Table 2 below. They show that ML-7significantly reduces the intra-aveolar accumulation of immune cells.TABLE 2 Effect of ML-7 on the level of accumulation of immune cells inbronchoalveolar lavage fluid induced by intratracheal instillation oftaurocholate (5 mM/rat) (means ± SD; n = 8) Control Taurocholate ML-7 +Taurocholate Leucocytes 4032 ± 919 35200 ± 12708* 4160 ± 573 Macrophages3419 ± 762 33288 ± 15531* 3585 ± 398 Lymphocytes 166 ± 68 3654 ± 2443*101 ± 44 Neutrophils  445 ± 113 5732 ± 2279*  473 ± 216Control: 20 min intratracheal infusion of sterile water; 5 mMtaurocholate intratracheal infusion: ML-7 (1 mg/kg/12 h, 36 h IP) +taurocholate*p < 0.001 significantly different from controls

Example 3 Reduction of the Bronchial Inflammatory Response by PD-98059

This example shows that PD-98059 (MEK1 kinase inhibitor) reduces theintra-aveolar accumulation of immune cells associated with opening oftight junctions, induced by intratracheal infusion of taurocholate(Table 3).

For this experiment, three groups of 8 male Wistar rats (250-300 g) weregiven either PD-98059 by the IP route (1 mg/kg/12 h, 36 h) or thevehicle alone (DMSO). One hour after the last administration, underurethane anesthesia (25 mg/kg IP), a slow intratracheal infusion of 200μl of physiologic serum containing (2 groups) or not containing (controlgroup) taurocholate (5 mM/rat) was given.

Bronchoalveolar lavage (BAL) was performed two hours after theintratracheal infusion of taurocholate or the vehicle. TABLE 3 Effect ofPD-98059 on the level of accumulation of immune cells in bronchoalveolarlavage fluid induced by intratracheal infusion of taurocholate (5mM/rat) (means ± SEM; n = 8). Results expressed as number of cells/mm³BAL. Control Taurocholate PD-98059 + taurocholate Leucocytes 5040 ± 62856637 ± 9791* 21424 ± 3164*# Macrophages 4582 ± 586 40234 ± 5799* 17956± 2465*# Lymphocytes 136 ± 31 4251 ± 940*  774 ± 183*# Neutrophils 320 ±62 11769 ± 5787* 2652 ± 600*# (Eosinophils) 0 602 ± 173  27 ± 27*#Control: sterile 0.9% NaCl 20 min intratracheal infusion; 5 mMtaurocholate per rat intratracheal infusion; PD-98059 (1 mg/kg/12 h, 36h) + taurocholate.*p < 0.05 significantly different from controls.#p < 0.05 significantly different from taurocholate values.

Example 4 ML7 Inhibits the Increase in Pulmonary Permeability Induced byPseudomonas aeruginosa LPS in the Rat.

This example shows that ML-7 (inhibitor of myosin light chain kinase,MLCK) significantly inhibits the increase in pulmonary permeabilityinduced by intratracheal infusion of Pseudomonas aeruginosalipopolysaccharide (LPS).

Pulmonary permeability was measured by means of a tracer, ¹²⁵I -labelledhuman serum albumin which, after intratracheal infusion, was determinedin urine, plasma, lung tissue and bronchoalveolar lavage fluid.

For this experiment, three groups of 6 male Wistar rats (200-225 g) werepretreated either with ML-7 by the IP route (3 mg/kg then 1 mg/kg 3times a day for 48 h) or the vehicle (ethanol 10%). One hour after thenext-to-last administration of ML-7, under urethane anesthesia (25 mg/kgIP), a slow intratracheal infusion of 150 μl of an iso-osmolar solution(5% bovine albumin+PBS) containing the tracer and containing (2 groups)or not containing (control group) LPS (1 μg/rat) was given.

Four hours after the intratracheal infusion, urine, blood,bronchoalveolar lavage (BAL) and lungs were harvested and ¹²⁵Iradioactivity was measured on each sample.

The results show that LPS decreases radioactivity measured in BALwhereas, at the same time, in comparison with controls, these levels aresignificantly higher in the lung. This increase in pulmonarypermeability is inhibited by pretreating the animals with ML-7. In fact,radioactivity levels in ML-7-treated animals were similar to controls inboth BAL and lung (FIG. 1).

In the three groups of animals, no differences in plasma radioactivitywere observed.

These results show that under such experimental conditions—which can beprojected to asthma and other allergic respiratory pathologies—ML-7 doesnot present effect on vascular endothelial cells which can modify theendothelium permeability, its tone (vasodilation) and thus generally onits pulmonary circulating hemodynamics (output, oxygenation i.e. healthgain in terms of hypoxia).

Furthermore, no traces of radioactivity were detected in the urine.

In conclusion, this example shows that Pseudomonas aeruginosa LPSincreases paracellular permeability in the lung, promoting theaccumulation of immune cells therein and that this effect can beprevented by treatment with an inhibitor of MLCK.

Example 5 ML-7 Reduces the Bronchial Inflammatory Response Induced byPseudomonas aeruginosa LPS in the Rat.

This example completes the previous example and demonstrates that ML-7reduces the bronchial inflammatory response induced by Pseudomonasaeruginosa LPS administered by intratracheal infusion. ML-7 therebyreduces the accumulation of immune cells in the alveoles.

For this experiment, three groups of 7 male Wistar rats (200-225 g) wereused. Animals were pretreated with either ML-7 by the IP route (3 mg/kgthen 1 mg/kg, 3 times a day for 48 h) or with the vehicle (ethanol 10%).One hour after the next-to-last ML-7 administration, under urethaneanesthesia (25 mg/kg IP), a slow intratracheal infusion of 150 μl of aniso-osmolar solution (5% bovine albumin+PBS) containing (2 groups) ornot containing (control group) LPS (1 μg/rat) was given. Four hoursafter the intratracheal infusion, bronchoalveolar lavage was performed.

The results show that LPS increases the levels of immune cells presentin BAL and more particularly that of polynuclear neutrophils. Thisincrease in the number of neutrophils is reduced by pretreating theanimals with ML-7 (Table 4). TABLE 4 Effect of ML7 on the level ofaccumulation of immune cells in bronchoalveolar lavage fluid induced byintratracheal infusion of LPS (1 μg/rat) (means ± SEM; n = 7). Resultsexpressed as the number of cells/mm³ BAL. Control LPS ML-7 + LPSLeucocytes 5883 ± 961 37968 ± 6912* 17243 ± 2956*# Macrophages 4760 ±738 16670 ± 3060* 10708 ± 1713*  Lymphocytes 188 ± 70 2253 ± 905* 902 ±375  Neutrophils  934 ± 707 19045 ± 4892*  6587 ± 1749*#Control: 5% bovine albumin + PBS 20 min intratracheal infusion; LPS 1μg/rat intratracheal infusion; ML-7 (3 mg/kg then 1 mg/kg 3 times a dayfor 48 h IP) + LPS*p < 0.05 significantly different from controls.#p < 0.05 significantly different from LPS values.

Exemple 6 LPS Stimulates Phosphorylation of Myosin Light Chain in HumanBronchial Epithelial Cells.

Materials: The human cell line NCI-H292 was obtained from the AmericanType Culture Collection (Manassas, Va.). Reagents, RPMI 1640 medium,fetal calf serum and other cell culture reagents were from Gibco, theprotease inhibitor cocktail from Roche (1697498) and LPS (E. coliSO₅₅:B5) and other reagents from Sigma.

Cell culture: NCI-H292 cells were cultured in RPMI 1640 mediumsupplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/mlstreptomycin and 10% fetal calf serum. Cells were grown at 37° C. in ahumidified 5% CO₂ atmosphere and subcultured twice a week. Cells wereseeded into 6-well plates at 5.10³ cells per well. At confluence, cellswere incubated in RPMI 1640 containing 0.1% bovine serum albumin (BSA)overnight. Cells were then washed with BSA-free RPMI 1640 and exposed toLPS (2 μg/ml) or physiologic serum as control (0.9% NaCl) for periodsranging from 30 min to 24 hours.

Western Blot: For this analysis, cells were lysed at different timesafter LPS treatment with RIPA buffer (1% Triton, 150 mM NaCl, 1 mM EDTA,10 mM Tris pH 7.4 and protease inhibitor cocktail at the supplier'srecommended concentration). The phosphorylated form of the myosin lightchain (p-MLC) and the non-phosphorylated form of the myosin light chain(MLC-20) were detected in the cells at 30 min, 1, 2 and 3 hours ofexposure to LPS as well as at longer periods (6, 12 and 24 hours).Proteins from LPS-treated NCI-H292 cells were separated by SDS-PAGE on a15% polyacrylamide gel and electrophoretically transferred to anitrocellulose membrane in 25 mM Tris-amino, 192 mM glycine and 20%methanol. Immunoprecipitation was with a goat anti-human phosphorylatedmyosin antibody diluted 1/500 (Santa Cruz Biotechnology Inc.) or a mousemonoclonal anti-human myosin antibody (light chains 20 K, Sigma-Aldrich,Inc.) diluted 1/1000, for detection of p-MLC and MLC-20, respectively.Peroxidase-recombinant protein G was used at 1/1000 dilution assecondary antibody. The immunolabelled bands were revealed byfluorography with ECL reagent (Enhanced chemiluminescence, Pierce,Perbio Science, Inc.).

Results: Compared to controls (physiologic serum), a maximalquantitative increase in the expression of the phosphorylated form p-MLCwas observed in LPS-treated cells after 30 min and 1 h of exposure (FIG.1), with a return to control values after 6 hours. Expression of thenon-phosphorylated form MLC-20 was low after 30 min and 1 h of LPStreatment and gradually increased over time (FIG. 1: Western blots).

Conclusions: Treatment of human bronchial cells with LPS leads to arapid increase (30 min to 1 hour) in the phosphorylation of myosin lightchains which comprise the cytoskeleton of these cells. Thisphosphorylation reflects the contraction of the cytoskeleton and theopening of tight junctions, thereby favoring the penetration ofallergens and the accumulation of immune cells in the bronchi.

Example 7 In Vitro Experimental Protocol for Determining the PlasmaActive Concentrations of Test Compounds and Able to Act

In vitro screening of pharmaceutical formulations can be an effectiveand profitable method to identify the lead candidate prior to in vivotesting. Cells form tight junctions which inhibit the passage of lowmolecular weight substances in solution and the flow of electricalcurrent, making it possible to use transepithelial resistance (TER) as acorrelate of permeability.

Measurement of electrical resistance: Human lung cells grow toconfluence on porous cell culture membrane inserts and their electricalresistance is measured with an electrical resistance device. An insertwithout a cell monolayer serves as control for baseline resistance andinserts with a confluent cell monolayer treated with PBS serve ascontrols. Pseudomonas aeruginosa LPS is added at concentrations of 1.0ng/ml to 10 μl/ml followed by incubation for 6 hours at 37° C.Transepithelial electrical resistance (ohms×centimeter squared) iscalculated by the following formula: (TER_(sample)−TER_(control))×area.Pseudomonas aeriginosa LPS decreases electrical resistance in adose-dependent manner. The value obtained corresponding to the maximumdose inducing a response with the maximum reduction in TER is considereda 100% response and is reported for the test compounds.

Test compounds: Using the same protocol with the maximum LPS dose (100%response), the test compounds are incubated 1 hour beforehand atconcentrations of 10 μM to 500 μM. The doses of test compound chosen forfuture tests or studies are the concentrations which cause a 50%reversion of the maximum LPS-induced decrease in TER.

This test may be employed to evaluate the 50% inhibitory effect of testcompounds on the decrease in TER following LPS treatment of cellmonolayers of human bronchial epithelial cells (NCI-H292).

1-12. (canceled).
 13. Method for the treatment of a respiratorypathology comprising administering to a subject in need thereof acompound that inhibits the contraction of the myosin light chain ofpulmonary epithelial cells.
 14. Method according to claim 13, whereinthe respiratory pathology is any respiratory pathology with theexception of hypoxia induced by said respiratory disease.
 15. Methodaccording to claim 13, wherein the compound inhibits the phosphorylationof the myosin light chain.
 16. Method according to claim 13, wherein thecompound is an inhibitor of MLCK.
 17. Method according to claim 13,wherein the compound is ML-7.
 18. Method according to claim 13, whereinthe compound is an activator of the myosin phosphatase.
 19. Methodaccording to claim 13, wherein the compound reduces paracellularpermeability of the pulmonary epithelium, without presenting asignificant effect on the vascular endothelium permeability.
 20. Methodaccording to claim 13, for reducing sensitization to allergens insubjects presenting with or sensitive to respiratory diseases. 21.Method according to claim 13, for the treatment of allergies, asthma orobstructive diseases.
 22. Method according to claim 13, for reducingtransepithelial migration of immune cells and accumulation of immunecells in the lung of subjects with a respiratory pathology.
 23. Methodaccording to claim 13, wherein the compound is administered by the oralroute or by inhalation.
 24. Method according to claim 13, wherein thecompound is used in association with another active agent selected inthe group consisting in □2-agonists, anticholinergics, corticosteroidsand anti-leukotrienes, in view of a combined use, separate use or spreadout over time.